Chromatographic and electrophoretic techniques are well known in the art as means for separating components (solutes) present in a mixture. These techniques are particularly useful in the chemical and biotechnological arts. True chromatography describes the separation of solutes according to their different partitioning between two (or three) phases. The phases generally are solid and liquid, and solute partitioning results in their differing mobilities through a layer of solid particles (matrix) in the presence of a flowing phase. Solute transfer through the layer may be along a pressure gradient, generally referred to as "liquid chromatography". In contrast, electrophoretic systems separate solutes on the basis of their electrophoretic mobility, isoelectric point, and/or differential migration through a size discriminating matrix. Solute transfer in these systems is driven by a voltage gradient from an applied electric field, e.g., "electrophoresis".
Chromatographic matrices can separate components by any of a number of criteria, including size, electrical charge, hydrophobic interactions and/or specific affinity for the matrix or binding sites thereon. Because the components in the mixture will vary in their affinity for the matrix, their partitioning as they pass through the matrix separates the components so that they exit the matrix sequentially, separated temporally and spacially. Determination of the location of the various separated components, or of a given component of interest within the sequence, generally is achieved by collecting the fluid phase exiting the matrix (effluent stream) as a series of fractions and sampling these fractions to identify their contents by any of a number of means known in the art.
Resolution of the various components in the mixture depends on several considerations, chief among them being the partitioning ability of the matrix and the system's theoretical plate height and plate number (see infra). In general, a large surface area-to-volume ratio is desired. Matrices for liquid chromatography systems typically are housed in cylindrical chromatography systems known as columns. In electrophoresis systems, high resolution also demands efficient removal of the heat generated by the applied electric field. Capillary electrophoresis, or other electrophoretic modules which provide a large surface area-to-volume ratio, dissipate Joule heat well, allowing rapid analysis without significant loss of resolution.
The need to monitor a product's status during its synthesis or purification is well known in the art. Status monitoring is particularly important in multi-step preparative protocols. Frequently, the identity and, often, quality of a product in a mixture must be determined at each step. Product monitoring also may be used as part of a feedback system to adjust process parameters. Generally, identification is determined using a previously established criterion for identification, for example, a characteristic absorbance measured at a given wavelength. If the product of interest is a protein, identification also may be by molecular weight, activity, and/or immunoaffinity.
Unfortunately, product and/or process monitoring may be undesirably time-consuming, particularly when multiple samplings are required, as during a multi-step preparation. The time limitation of monitoring is of particular concern in industrial or other large scale preparations. To be useful, the monitor step should be rapid, adaptable, and repeatable. Accordingly, there exists a need for a method of rapidly identifying the presence and location of a molecule of interest during any preparative protocol.
It is therefore an object of this invention to provide a method and apparatus for the rapid, on-line identification of molecular products during a preparative protocol, such as during their synthesis or purification. Another object is to provide a method and apparatus for rapidly identifying the presence and location of a solute in a chromatography effluent. Another object is to provide a method for rapidly detecting the concentration of a product in an effluent stream. Still another object is to provide a method for rapidly assessing the success of a purification or separation protocol. These and other objects and features of the invention will be apparent from the drawing, description, and claims which follow.